The majority of complex congenital heart defects occur in individuals who are also afflicted by laterality disease, reflecting the extreme susceptibility of the developing heart to disturbances in left-right (LR) patterning processes. The objective of this proposal is to identify inductive mechanisms that generate the LR body plan and to elucidate how cardiac cells use this positional "blueprint" to develop LR asymmetries. With focus on receptors for the transforming growth factor-Beta superfamily, we have found that two Activin-Like Kinases--ALK2 and ALK4--are both necessary and sufficient for LR development of the heart (and other organs) in embryos of the frog, Xenopus Iaevis. Taking advantage of the range of experimental manipulations that is uniquely possible in Xenopus, we will test the hypothesis that ALK2 and ALK4 modulate cardiac LR development by playing pivotal roles in LR axis determination. Using both biochemical and loss-of-function analyses, Aim 1 will identify cell-cell signals important for LR axis formation by defining ligands for the ALK pathways. Using classic transplantation and co-culture assays, Aim 2 will position ALK pathways in the context of cell-cell interactions already known to be important for LR patterning by determining whether ALKs relay positional signals to the midline. Because many of the molecules involved in midline specification and other LR relay interactions are not known, Aim 3 will fill in some of these gaps by identifying genes that function upstream, downstream, or within ALK pathways. This will be accomplished by performing a high throughput expression cloning screen in which new laterality genes already have been, and will continue to be, identified by the applicant. Using in vivo lineage labeling, Aim 4 will address how cardiac cells utilize LR patterning information by determining whether ALK signaling culminates in specific asymmetric cell contributions from the paired left and right heart fields to different segments of the "straight" heart tube. Together, our results will contribute new knowledge that is necessary to accelerate progress in the intersecting fields of embryonic heart formation and vertebrate LR development. The long-term significance of the work will be to identify genes and inductive processes that may result in congenital cardiac (and other) LR defects if affected by genetic or environmental perturbations.